The present invention, in some embodiments thereof, relates to the use of the reductive glycine pathway for the generation of formatotrophic and autotrophic microorganisms.
The concept of biorefineries has become a wide spread notion in the last decade. It relies on the premise that living organisms can and should be used to supply the increasing demand by humanity for specialized chemicals, including fuels, solvents, plastics, pharmaceuticals, etc. Today, most of these chemicals are derived, directly or indirectly, from fissile carbons. However, with the imminent depletion of these fossil carbons and the increase in atmospheric CO2 it has become essential to find alternative sources for these important materials.
The suggested feedstocks for most of the proposed biorefineries are simple sugars, starch, or lignocellulosic biomass. While the latter alternative has an apparent advantage over the former by not-competing with human consumption needs, it still presents numerous difficulties, including a problematic fermentation technology and feedstock availability and transportation. A fascinating alternative feedstock would be electric current. Electrons can be shuttled from an electrode to living cells, providing the necessary reducing equivalents and energy to support autotrophic growth and electrosynthesis of desired commodities (1-5). Since electricity is widely available, microbial electrosynthesis can be spatially and temporally decoupled from energy production and can take place at any convenient location and time.
Microbial electrosynthesis can be especially useful for the renewable energy market. One major drawback of most renewable energy sources, including solar, wind, hydro and nuclear, is that they are hard to store in a convenient way. Microbial electrosynthesis of fuels can thus serve to address this problem efficiently, converting electrical energy to kinetically stable chemical bonds.
Several methods of transferring reducing equivalents from an electrode to living cells were suggested and applied (reviewed in 1-5). Molecular hydrogen is one of the earliest electron carriers used in this manner since water electrolysis is a relatively mature technology that can support efficient hydrogen production at high current density. However, the use of hydrogen suffers from numerous problems including its low solubility and the risk of explosion. Moreover, the hydrogenase enzymes that transfer hydrogen's electrons to the cellular carriers are generally complex, oxygen sensitive proteins, which are hard to recombinantly express and consume a significant fraction of the cell resources. As an alternative to molecular hydrogen, several inorganic compounds, such as ferric ion or nitrate, can serve as electron shuttles, supporting electricity-dependent cultivation (5). However, since the reduction potentials of these compounds are considerably higher than that of NAD(P)H, reverse electron flow must take place during growth on these substrates, limiting electrosynthesis to specific organisms which are less suitable to industrial use. A further option is direct electron transfer from the cathode to the microbes. While several advantages of this option were proposed (reviewed in 2-5) this approach is limited to a small group of organisms or requires complex adaption of others.
As an alternative to all of the above methods, CO2 can be directly reduced at the cathode (the electrons are derived from water splitting at the anode) (6), providing organic compounds that can be used by living cells as a source of reducing equivalents, energy and even carbon. A diverse group of compounds can be produced in this manner (6-9). The production of simple alcohols, such as methanol, ethanol and propanol, hydrocarbons, such as methane and ethylene, or acids with more than one carbon, such as acetic acid and oxalic acid, has the advantage of supplying microbes with compounds relatively simple to metabolize and/or being rich in reducing equivalents. However, the electrocatalytic production of all of these compounds is generally inefficient (not specific to a single product and/or requiring high overpotential), requiring costly catalysts and/or supporting low current density (reviewed in 6, 7). In contrast, there are two compounds that can be produced by direct reduction of CO2 at relatively high efficiency (although lower than that of molecular hydrogen) and an acceptable current density: carbon monoxide and formic acid (6-13). Since carbon monoxide is a toxic and flammable gas with low solubility, formic acid, being readily soluble and of low toxicity, is a preferred mediator of electrons. In fact, a formate-based economy was recently proposed as an alternative to the hydrogen-based economy or methanol-based economy concepts (14-17).
Various methylotrophic organisms can grow on formate as a sole carbon, electron and energy source (18-21). Such organisms can be used for formate-dependent microbial electrosynthesis (19, 22). However, as compared to model organisms extensively used in the bioindustry, such as S. cerevisiae or E. coli, the metabolism of these microbes is far less understood, their bulk cultivation is limited and their genetic manipulation is considerably less optimized. As a consequence, biotechnological usage of these natural methylotrophs is usually limited to the production of simple products.